PROJECT ABSTRACT (provided by applicant): Tsg101, a component of ESCRT-I (endosomal sorting complex required for transport-I), is required for budding of infectious HIV and several other human pathogens. A proline-rich sequence, PTAP, in the viral-encoded structural precursor polyprotein, Gag, recruits the cellular factor to virus assembly sites on the plasma membrane by interacting with a PTAP-binding pocket in the Tsg101 N-terminal UEV [ubiquitin (Ub) E2 variant] domain. The UEV domain also binds Ub, however, the role of Ub in budding and the relationship of UEV Ub-binding to the PTAP-mediated recruitment of the ESCRT machinery is not clear. Recently, using small molecules identified by high-throughput screening of a library for known drugs capable of binding the UEV, we found agents that inhibit HIV-1 budding by disrupting Ub- binding at the known pocket in the UEV without disturbing the PTAP-binding pocket. Our NMR studies identified an additional Ub-binding site in the Tsg101 UEV domain and demonstrate that the domain is capable of binding di-Ub moieties previously shown to participate in both endocytic trafficking and budding. This application proposes to use the agents, mutagenesis and structural analysis to define the mechanism by which the original and newly identified UEV Ub-binding sites facilitate the budding process through achievement of 3 goals. The goal of AIM 1 is structure- function analysis of the UEV-Ub complex employing site-directed mutagenesis and small molecule probes. As we and others find that, while necessary, PT/SAP-mediated recruitment of Tsg101 alone is not sufficient for budding, the goal of AIM 2 is to test the hypothesis that the Tsg101 Ub- binding function is required in conjunction with the PTAP-binding function for efficient recruitment of the protein to the plasma membrane. The goal of AIM 3 is to determine whether the Tsg101 Ub-binding function is important for trafficking pathways where Tsg101 is not directly engaged by a viral structural protein, i.e., pathways facilitated by the ESCRT adaptors Alix and Nedd4. It is well-established that HIV utilizes alternative budding pathways when Gag interaction with Tsg101 is disrupted: It is therefore important to understand if the required Tsg101-Ub binding function is redundant. Collectively, the proposed studies will provide mechanistic understanding of how Tsg101 facilitates HIV-1 Gag trafficking and virus budding and possibly also reveal new targets for anti-viral drug development.